Our previous discovery of the structure of the turnip crinkle virus tRNA-like translational enhancer (TCV TSS) has permitted us to pursue the use of a relatively new technique for understanding the structural characteristics of an RNA when optical tweezers are applied to pull the molecular structure apart. Essentially a force is applied to the 5 and 3 prime ends of the molecule, which is then monitored. Force changes are then correlated with structural features. The pulling experiments, in collaboration with Anne Simon, were correlated to simulated steered molecular dynamics, which enabled the visualization of the unfolding events of the molecule as a function of the pulling speed and forces applied. These results shed light on the significant rearrangement that occurs in the translational enhancer tRNA-like structure as a result of RNA dependent RNA polymerase (RdRp) binding to this region to produce the negative strand of the virus.Some unexpected structural relationships were found that at least in part, showed how major rerangements occur in the TSS as a result of RdRp binding. Coarse-grained and explicit solvent techniques were used to elucidate the structural characteristics. This technique offers a unique methodology for understanding RNA structure and the characteristics of various RNA motifs found in the structure. ---We have also pursued, in collaboration with Shuo Gu, a comprehensive examination of potential RNA-RNA interactions that are found across 4 different cell lines. MySeq reads were examined and correlated with computational bioinformatic analysis of potential interactions. The prevalence or lack thereof was determined to enable a better understanding of how cellular RNA interacts with its cellular environment. Interestingly the major finding was that there are very few RNA-RNA interactions found across the 4 cell lines, thus indicating the such interactions are avoided. The number of interactions went up significantly when proteins were removed from the lysates and the sequences re-annealed. Cells presumably avoid such interactions to prevent activation of innate immune responses, which are normally reserved for viruses. ---The functionality of Drosha in cellular systems is important for understanding the processing of microRNAs and how they relate to normal cellular activity as well as diseases such as cancer. In another collaboration with Shuo Gu the relationship of Drosha targeted stem-loop structures and the type of microRNA isforms that are produced was examined. Experimental and computational approaches were applied to determine these relationships. Results indicate that bent, distorted and/or flexible structures in the targeted Drosha stem seem to facilitate the production of alternate forms of microRNA. Structural predictions and experimental results were compared and correlated. ---A collaboration with Esta Sterneck's laboratory is ongoing. Her lab investigates cell signaling pathways involved in breast and glioblastoma tumorigenesis with a focus on the transcription factor CCAAT/enhancer binding protein delta (CEBPD) using in vitro cell culture and in vivo mouse model systems. Using a transgenic mouse model of breast cancer, her group has shown that CEBPD exhibits a dual role in mammary tumorigenesis. On the one hand, CEBPD prevents tumor multiplicity and on the other hand, CEBPD promotes distant lung metastases. In addition, CEBPD promotes stem-like cancer cells, which have been implicated in tumor metastasis and treatment resistance, in breast and glioblastoma tumor cells through regulation of various signaling pathways and stemness. In addition, strategies for targeting the message of CEBPD are necessary to down regulate CEBPD-mediated tumor progression signaling. As a tie-in to our nanobiology project, our laboratory is developing approaches for RNAi therapeutics to knock down the CEBPD mRNA by delivering strategically designed RNA nanostructures as their own entities or in combination with lipid carriers.Initial results with our lipid-based carriers look promising and we are currently progressing to the use of mouse models for further verification.--- Due to the need to robustly produce large quantities of RNA of various lengths and for various purposes, a collaboration with Mikhail Kashlev has been established to accomplish this purpose using a common enzyme, E. coli RNA polymerase. This need has arisen, in part, due to the establishment of the RNA Biology Laboratory, potential needs as a therapeutic, as well as existing requirements of the NIH community. Currently, scaled production costs are high when ordering from companies that specialize in production. Costs become even more prohibitive when modified bases need to be included at specific positions within the RNA. Typically, chemical synthesis techniques are limited to under 100 bases and a common method of using RNA T7 polymerase, which may be useful for certain sequences does not perform well for all sequence compositions when modified bases are required. The use of E. coli RNA polymerase provides a potential avenue for the production of RNA for a variety of needs. ---The prediction of RNA secondary and 3D structures containing non-canonical base pair interactions is a difficult and important problem that needs better algorithms. We are developing a set of computational algorithms based on Bayesian and neural network methodologies to enable the prediction of canonical and more importantly non-canonical base pair interactions in RNA. A large database has been compiled containing a multitude of structures including the non-canonical base pair interactions. The algorithms have shown significant utility, enabling the prediction of complex motifs at the secondary structure level. These results are then being used in conjunction with an RNA 3D structure generation program, which enables the prediction of 3D RNA structures that incorporate the complex non-canonical interactions. This set of algorithms are also being applied to the prediction of multi-sequence RNA nano-assemblies. ---In another project in collaboration with Mikhail Kashlev we are developing a methodology using a neural network approach to determine transcriptional pause sites in bacterial cells. Sequence reads are being produced by a method called RNet-seq (a variation of Net-seq) to define pause sites. The neural net being developed using these reads discriminates between these types of sites in various bacterial sequences. ---Another project in collaboration with Stuart Le Grice involves the development of a computational approach to determined binding sites and affinities of small molecules targeting various RNA structural motifs. The goal of this project is to aid in the screening of small molecules for their potential to be therapeutically beneficial in targeting viral RNAs or cancer causing genes. The small molecules are initially derived from sets found my binding to experimental screening methods using small molecule microarrays. ---An algorithm, RiboSketch, has been developed for the depiction of nucleic acid secondary structures which may contain multiple strands consisting of RNA or DNA. Layout algorithms, comprehensive editing capabilities, and a multitude of simulation modes are available within the system.Iinteractive features allow RiboSketch to create publication quality diagrams for structures with a wide range of composition, size, and complexity. The program may be run in any web browser without the need for installation, or as a standalone Java application.